2.3 DNA amplification
Two primer pairs were used, each amplifying either the Unionida or the Venerida (Prié et al., 2020; presented in Table 1). Each DNA extract was amplified in twelve replicates per primer pair, including all the extraction negative controls and the PCR negative controls.
The amplification mixture contained 1 U of AmpliTaq Gold Polymerase (Applied Biosystems), 1x PCR Gold buffer containing 2 mM of MgCl2, 0.2 mM of each dNTPs, 0.5 μM of each tagged forward and reverse primers and 0.2 mg/mL of bovine serum albumin (BSA, Roche Diagnostics). The final volume was 25 μL including 3 μL of eDNA extraction. All amplification underwent the same PCR parameters (initial denaturation for 3 min at 95°C, 50 cycles of 30 s at 95°C, 30 s at 50°C and 30 s at 72°C, and final elongation at 72°C for 5 min). Forward and reverse primers were 5‘-labeled with identical eight nucleotides tags to enable the subsequent assignment of sequences to their respective sample. Each tag had at least four differences with other tags to properly assign reads to samples. A total of 65 negative extraction controls and 35 PCR negatives controls (ultrapure water) were also included in the experiment to monitor possible contaminations. Pooled PCR products were purified using a MinElute PCR purification kit (Qiagen), with elution in 15 μl buffer.